miRNA-REGULATED DIFFERENTIATION-DEPENDENT SELF-DELETING CASSETTE

ABSTRACT

Targeting constructs and methods of using them are provided for differentiation-dependent modification of nucleic acid sequences in cells and in non-human animals. Targeting constructs comprising a promoter operably linked to a recombinase are provided, wherein the promoter drives transcription of the recombinase in an differentiated cell but not an undifferentiated cell. Promoters include Blimp1, Prm1, Gata6, Gata4, Igf2, Lhx2, Lhx5, and Pax3. Targeting constructs with a cassette flanked on both sides by recombinase sites can be removed using a recombinase gene operably linked to a 3′-UTR that comprises a recognition site for an miRNA that is transcribed in undifferentiated cells but not in differentiated cells. The constructs may be included in targeting vectors, and can be used to automatically modify or excise a selection cassette from an ES cell, a non-human embryo, or a non-human animal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 USC §119(e) of U.S.Provisional Patent Application Ser. No. 61/233,974, filed 14 Aug. 2009.

FIELD OF INVENTION

The invention relates to nucleic acid constructs comprising cassettesfor deleting nucleic acid sequences from genetically modified cells andanimals, in particular for use in connection with targeting vectors inorder to delete nucleic acid sequences introduced into a cell or ananimal using a targeting vector.

BACKGROUND

Targeted gene modification in the mouse (commonly referred to asknockout mouse technology because the goal of many of the modificationsis to abolish, or knock out, target gene function) is the most effectivemethod for discovery of mammalian gene function in live animals and forcreating genetic models of human disease. Knockout mouse creationtypically begins by introducing a targeting vector into mouse embryonicstem (ES) cells. The targeting vector is a linear piece of DNAcomprising a selection or marker gene (e.g., for drug selection) flankedby mouse DNA sequences—the so-called homology arms—that are similar oridentical to the sequences at the target gene and which promoteintegration into the genomic DNA at the target gene locus by homologousrecombination. To create a mouse with an engineered geneticmodification, targeted ES cells are introduced into mouse embryos, forexample pre-morula stage (e.g., 8-cell stage) or blastocyst stageembryos, and then the embryos are implanted in the uterus of a surrogatemother (e.g., a pseudopregnant mouse) that will give birth to pups thatare partially or fully derived from the genetically modified ES cells.After growing to sexual maturity and breeding with wild type mice someof the pups will transmit the modified gene to their progeny, which willbe heterozygous for the mutation. Interbreeding of heterozygous micewill produce progeny that are homozygous for the modified allele and arecommonly referred to as knockout mice.

The initial step of creating gene-targeted ES cells is a rare event.Only a small portion of ES cells exposed to the targeting vector willincorporate the vector into their genomes, and only a small fraction ofsuch cells will undergo accurate homologous recombination at the targetlocus to create the intended modified allele. To enrich for ES cellsthat have incorporated the targeting vector into their genomes, thetargeting vector typically includes a gene or sequence that encodes aprotein that imparts resistance to a drug that would otherwise kill anES cell. The drug resistance gene is referred to as a selectable markerbecause in the presence of the drug, ES cells that have incorporated andexpress the resistance gene will survive, that is, be selected, and formclonal colonies, whereas those that do not express the resistance genewill perish. Such a selectable marker is typically present in aselection cassette, which typically includes nucleic acid sequences thatwill allow for expression of the selectable marker. Molecular assays ondrug-resistant ES cell colonies identify those rare clones in whichhomologous recombination between the targeting vector and the targetgene results in the intended modified sequence (e.g., the intendedmodified allele).

After selection of drug-resistant clones, the selection cassettetypically serves no further function for the modified allele. Ideallythe cassette should be removed, leaving an allele with only the intendedgenetic modification, because the selection cassette might interferewith the expression a neighboring gene such as a reporter gene, which isoften incorporated adjacent to the selectable marker in many knockoutalleles, or might interfere with a nearby endogenous gene (see, e.g.,Olsen et al. (1996) Know Your Neighbors: Three Phenotypes of theMyogenic bHLH Gene MRF4. Cell 85:1-4; Strathdee et al. (2006) Expressionof Transgenes Targeted to the Gt(ROSA)26S or Locus Is OrientationDependent, PloS ONE 1(1):e4). Either event can confound theinterpretation of the phenotype of the modified allele. For thesereasons selectable markers in knockout alleles are usually flanked byrecognition sites for site-specific recombinase enzymes, for example,loxP sites, which are recognized by the Cre recombinase (see, e.g.,Dymecki (1999) Site-specific recombination in cells and mice, in GeneTargeting: A Practical Approach, 2d Ed., 37-99). A typical selectioncassette comprises a promoter that is active in ES cells linked to thecoding sequence of an enzyme, such as neomycin phosphotransferase, hatimparts resistance to a drug, such as G418, followed by apolyadenylation signal, which promotes transcription termination and 3′end formation and polyadenylation of the transcribed mRNA. This entireunit is flanked by recombinase recognition sites oriented to promotedeletion of the selection cassette upon the action of the cognaterecombinase.

Recombinase-catalyzed removal of the selection cassette from theknockout allele is typically achieved either in the gene-targeted EScells by transient expression of an introduced plasmid carrying therecombinase gene or by breeding mice derived from the targeted ES cellswith mice that carry a transgenic insertion of the recombinase gene.Either method has its drawbacks. Selection cassette excision bytransient transfection of ES cells is not 100% efficient. Incompleteexcision necessitates isolating multiple subclones that must be screenedfor loss of the selectable marker, a process that can take one to twomonths and subject a targeted clone to high levels of recombinase and asecond round of electroporation and plating that can adversely affectthe targeted clone's ability to transmit the modified allele through thegermline. Consequently, the process might require repetition on multipletargeted clones to ensure the successful creation of knockout mice fromthe cassette-deleted clones.

The alternative approach of removing the selection cassette in micerequires even more effort. To achieve complete removal of the selectioncassette from all tissues and organs, mice that carry the knockoutallele must be bred to an effective general recombinase deletor strain.But even the best deletor strains are less than 100% efficient atpromoting cassette excision of all knockout alleles in all tissues.Therefore, progeny mice must be screened for correct recombinants inwhich the cassette has been excised. Because mice that appear to haveundergone successful cassette excision may still be mosaic (i.e.,cassette deletion was not complete in all cell and tissue types), asecond round of breeding is required to pass the cassette-excised allelethrough the germline and ensure the establishment of a mouse linecompletely devoid of the selectable marker. In addition to about sixmonths for two generations of breeding and the associated housing costs,this process may introduce undesired mixed strain backgrounds throughbreeding, which can make interpretation of the knockout phenotypedifficult.

Accordingly, there remains a need in the art for compositions andmethods for excising nucleic acid sequences in genetically modifiedcells and animals.

SUMMARY

Compositions and methods for excising nucleic acid sequences ingenetically modified cells and animals are provided, and, in particular,for excising nucleic acid sequences.

In one aspect, an expression construct is provided, wherein theexpression construct comprises a promoter operably linked to a geneencoding a site-specific recombinase (recombinase), wherein the promoterdrives transcription of the recombinase in differentiated cells, butdoes not drive transcription of the recombinase in undifferentiatedcells. Unifferentiated cells include ES cells, e.g., mouse ES cells.

In one embodiment, the expression construct further comprises aselection cassette, wherein the selection cassette is disposed between afirst recombinase recognition site (RRS) and a second RRS, wherein therecombinase recognizes both the first and the second RRS.

In one embodiment, the first and the second RRS are nonidentical. In oneembodiment, the first and the second RRS are independently selected froma loxp, lox511, lox2272, lox66, lox71, loxM2, lox5171, FRT, FRT11,FRT71, attp, att, FRT, or Dre site.

In one embodiment, the first and the second RRS are oriented so as todirect a deletion in the presence of the recombinase.

In one embodiment, the selection cassette comprises a gene that confersresistance to a drug.

In one aspect, a method for excising a selectable marker from a genomeis provided, comprising the step of allowing a cell to differentiate,wherein the cell comprises a selection cassette, wherein the selectioncassette is flanked 5′ and 3′ by site-specific recombinase recognitionsites (RRSs); and wherein the cell further comprises a promoter operablylinked to a gene encoding a recombinase that recognizes the RRSs,wherein the promoter drives transcription of the recombinase indifferentiated cells at least 10-fold higher than it drivestranscription of the recombinase in undifferentiated cells, whereinfollowing expression of the recombinase, the selection cassette isexcised.

In one embodiment, the promoter drives transcription in differentiatedcells about 20-, 30-, 40-, 50-, or 100-fold higher than it drivestranscription in undifferentiated cells. In one embodiment, the promoterdoes not substantially drive transcription in undifferentiated cells,but drives transcription in differentiated cells.

In one embodiment, expression of the recombinase in a culture of cellsmaintained under conditions sufficient to inhibit differentiation,occurs in no more than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or0.9% of the cells of the culture. In one embodiment, expression occursin no more than about 1, 2, 3, 4, or 5% of the cells of the culture.

In one embodiment, the promoter is selected from a Prm1 (aka, Prdm1),Blimp1, Gata6, Gata4, Igf2, Lhx2, Lhx5, Pax3. In a specific embodiment,the promoter is the Gata6 or Gata4 promoter. In another specificembodiment, the promoter is a Prm1 promoter. In another specificembodiment, the promoter is a Blimp1 promoter or fragment thereof, e.g.,a 1 kb or 2 kb fragment of a Blimp1 promoter.

In one embodiment, the cassette is on a separate nucleic acid moleculethan the recombinase gene. In one embodiment, the selection cassette andthe recombinase gene are on a single nucleic acid molecule. In aspecific embodiment, RSSs flank, 5′ and 3′, a nucleic acid sequence thatincludes the selection cassette and the recombinase gene, such thatafter the recombinase binds the RSSs, the recominase gene and theselection cassette are simultaneously excised.

In one embodiment, the selection cassette is on a first targeting vectorand the recombinase gene is on a second targeting vector, wherein thefirst and the second targeting vector each comprise mouse targetingarms.

In one embodiment, the selection cassette and the recombinase gene areboth on the same targeting vector. In one embodiment, the cassette andthe recombinase gene are each positioned between the same two RRSs. Inone embodiment, the RRSs are arranged so as to direct a deletion. In oneembodiment, the RRSs are nonidentical. In one embodiment, the RRSs areeach recognized by the same recombinase. In a specific embodiment, theRRSs are nonidentical, are recognized by the same recombinase, and areoriented to direct a deletion of the recombinase gene and the cassette.In a specific embodiment, the RRSs are identical and are oriented todirect a deletion of the recombinase gene and the cassette.

In a specific embodiment, the targeting vector comprises, from 5′ to 3′with respect to the direction of transcription, a reporter gene; a firstRRS; a selectable marker driven by a first promoter; a second promoterselected from a Prm1, Blimp1, Gata6 and Gata4 promoter, wherein thesecond promoter is operably linked to a sequence encoding a recombinase;and a second RRS; wherein the first and the second RRS are in the sameorientation (i.e., in an orientation that, in the presence of therecombinase, directs deletion of sequences flanked by the RRSs).

In one embodiment, allowing the cell to differentiate comprises removingor substantially removing from the presence of the cell a factor thatinhibits differentiation. In a specific embodiment, the factor isremoved by washing the cell or by dilution of the cell in a medium thatlacks the factor that inhibits differentiation. In one embodiment,allowing the cell to differentiate comprises exposing the cell to adifferentiation factor at a concentration that promotes differentiationof the cell.

In one aspect, a targeting vector is provided, wherein the targetingvector comprises (a) a selection cassette; and, (b) a promoter operablylinked to a gene encoding a recombinase; wherein the cassette is flanked5′ and 3′ by RRSs recognized by the recombinase, wherein the promoterdrives transcription of the recombinase in differentiated cells, but notin undifferentiated cells.

In one embodiment the targeting vector further comprises flankingtargeting arms, each of which are mouse or rat targeting arms.

In one embodiment, the targeting vector further comprises a reportergene. In one embodiment, the reporter gene is selected from thefollowing genes: luciferase, lacZ, green fluorescent protein (GFP),eGFP, CFP, YFP, eYFP, BFP, eBFP, DsRed, and MmGFP. In a specificembodiment, the reporter gene is a lacZ gene.

In one embodiment, expression of a selectable marker of the selectioncassette (e.g., neo^(r)) is driven by a promoter selected from a UbCpromoter, an hCMV promoter, an mCMV promoter, a CAGGS promoter, an EF1promoter, a Pgk1 promoter, a beta-actin promoter, and a ROSA26 promoter.

In one embodiment, the gene encoding the recombinase is driven by apromoter selected from the group consisting of the following promoters:a Prm1, Blimp1, Blimp1 (1 kb fragment), Blimp1 (2 kb fragment), Gata6,Gata4, Igf2, Lhx2, Lhx5, and Pax3. In a specific embodiment, thepromoter is the Gata6 or Gata4 promoter. In another specific embodiment,the promoter is a Prm1 promoter. In another specific embodiment, thepromoter is a Blimp1 promoter or fragment thereof, e.g., a 1 kb fragmentor 2 kb fragment as described herein.

In one embodiment, the recombinase is selected from the group consistingof the following recombinases: Cre, Flp (e.g., Flpe, Flpo), and Dre.

In one embodiment, the RRSs are independently selected from a loxp,lox511, lox2272, lox66, lox71, loxM2, lox5171, FRT, FRT11, FRT71, attp,att, FRT, or Dre site.

In one embodiment, the selection cassette comprises a selectable markerfrom the group consisting of the following genes: neomycinphosphotransferase (neo^(r)), hygromycin B phosphotransferase (hyg^(r)),puromycin-N-acetyltransferase (puro^(r)), blasticidin S deaminase(bsr^(r)), xanthine/guanine phosphoribosyl transferase (gpt), and Herpessimplex virus thymidine kinase (HSV-tk). In a specific embodiment, theselection cassette comprises a neo^(r) gene driven by a UbC promoter.

In one embodiment, the targeting vector comprises (a) a selectioncassette flanked 5′ and 3′ by a loxp site; and, (b) a Prm1, Blimp1,Gata6, Gata4, Igf2, Lhx2, Lhx5, or Pax3 promoter operably linked to agene encoding a Cre recombinase, wherein the Gata6, Gata4, Igf2, Lhx2,Lhx5, or Pax3 promoter drives transcription of the Cre recombinase indifferentiated cells, but does not drive transcription, or does notsubstantially drive transcription, in undifferentiated cells.

In one embodiment, the targeting vector comprises, from 5′ to 3′ withrespect to the direction of transcription of the targeted gene: (a) a 5′targeting arm; (b) a reporter gene; (c) a first RRS; (d) a selectioncassette; (e) a promoter operably linked to a nucleic acid sequenceencoding a recombinase; (f) a second RRS; and, (g) a 3′ targeting arm;wherein the promoter drives transcription of the recombinase gene indifferentiated cells, and does not drive transcription of therecombinase gene in undifferentiated cells or does not substantiallydrive transcription of the recombinase in undifferentiated cells.

In one aspect, a method for excising a nucleic acid sequence in agenetically modified non-human cell is provided, comprising a step ofallowing a cell to differentiate, wherein the cell comprises a selectioncassette flanked 5′ and 3′ by RRSs and further comprises a promoteroperably linked to a gene encoding a recombinase that recognizes theRRSs, further comprising a 3′-UTR of the recombinase gene, wherein the3′-UTR of the recombinase gene comprises a sequence recognized by anmiRNA that is active in an undifferentiated cell but is not active in adifferentiated cell, wherein following differentiation, the recombinasegene is transcribed and expressed such that the selection cassette isexcised.

In one embodiment, the miRNA is present in the undifferentiated cell ata level that inhibits or substantially inhibits expression or therecombinase gene; wherein the miRNA is absent in a differentiated cellor is present in a differentiated cell at a level that does not inhibit,or does not substantially inhibit, expression of the recombinase gene.

In one aspect, a targeting vector is provided, wherein the targetingvector comprises a nucleic acid sequence encoding a recombinase followedby a 3′-UTR, wherein the 3′-UTR comprises an miRNA recognition site,wherein the miRNA recognition site is recognized by an miRNA that isactive in undifferentiated cells and is not active in differentiatedcells.

In one aspect, a targeting vector is provided, wherein the targetingvector comprises, from 5′ to 3′ with respect to the direction oftranscription of the targeted gene: (a) a 5′ targeting arm; (b) areporter gene; (c) a first RRS; (d) a nucleic acid sequence encoding aselectable marker operably linked to a first promoter that drivesexpression of the marker; (e) a recombinase gene operably linked to asecond promoter; (g) a 3′-UTR comprising an miRNA recognition site,wherein the miRNA recognition site is recognized by an miRNA that isactive in undifferentiated cells and is not active in differentiatedcells; (h) a second RRS; and, (i) a 3′ targeting arm.

In one embodiment the miRNA recognition site recognizes an miRNA of themiR-290 cluster. In one embodiment, the miR-290 cluster member ismiR-292-3p, 290-3p, 291a-3p, 291 b-3p, 294, or 295; in a specificembodiment, the miRNA recognition site comprises a seed sequence of oneor more of the aforementioned miR-290 cluster members. In a specificembodiment, the miRNA recognition site recognizes an miRNA thatcomprises the seed sequence of miR-292-3p or miR-294.

In one embodiment, the miRNA recognition site recognizes an miRNA of themiR-302 cluster (miR-302a, 302b, 302c, 302d, and 367). In oneembodiment, the miR-302 cluster member is miR-302a, 302b, 302c, or 302d;in a specific embodiment, the miRNA recognition site comprises a seedsequence of one or more of the aforementioned miR-302 cluster members.

In one embodiment, the miRNA recognition site recognizes an miRNA of themiR-17 family (miR-17, miR-18a, miR-18b, miR-20a). In one embodiment,the miR-17 family member is miR-17, miR-18a, miR-18b, miR-20a; in aspecific embodiment, the miRNA recognition site comprises a seedsequence of one or more of miR-17, miR-18a, miR-18b, or miR-20a.

In one embodiment, the miRNA recognition site recognizes an miRNA of themiR-17-92 family (including miR-106 and miR-93). In one embodiment, thefamily member is miR-106a, miR-18a, miR-18b, miR-93, or miR-20a; in aspecific embodiment, the miRNA recognition site comprises a seedsequence of one or more of miR-106a, miR-18a, miR-18b, miR-93, ormiR-20a.

In one embodiment, the miRNA recognition site recognizes an miRNA whoseseed sequence (nucleotides 2 to 8 from the 5′ end) is identical or has 6out of 7 nucleotides of the seed sequence of an miRNA selected frommiR-292-3p, miR-290-3p, miR-291a-3p, miR-291b-3p, miR-294, miR-295,miR-302a, miR-302b, miR-302c, miR-302d, miR-367, miR-17, miR-18a,miR-18b, miR-20a, miR-106a, or miR-93. In one embodiment, the miRNArecognition site further comprises a sequence outside of the seedrecognition site, wherein the sequence outside of the seed recognitionsite is substantially complementary to the non-seed sequence of a miRNAselected from miR-292-3p, miR-290-3p, miR-291a-3p, miR-291b-3p, miR-294,miR-295, miR-302a, miR-302b, miR-302c, miR-302d, miR-367, miR-17,miR-18a, miR-18b, miR-20a, miR-106a, or miR-93. In a specificembodiment, the miRNA recognition site comprises a sequence outside ofthe seed recognition site has a complementarity of about 80%, 85%, 90%,or 95% with a non-seed sequence of a miRNA selected from miR-292-3p,miR-290-3p, miR-291a-3p, miR-291b-3p, miR-294, miR-295, miR-302a,miR-302b, miR-302c, miR-302d, miR-367, miR-17, miR-18a, miR-18b,miR-20a, miR-106a, or miR-93. In a specific embodiment, the non-seedsequence of the miRNA recognition site is perfectly complementary to anon-seed sequence of an miRNA selected from miR-292-3p, miR-290-3p,miR-291a-3p, miR-291b-3p, miR-294, miR-295, miR-302a, miR-302b,miR-302c, miR-302d, miR-367, miR-17, miR-18a, miR-18b, miR-20a,miR-106a, or miR-93.

In one embodiment, the reporter gene is selected from luciferase, lacZ,green fluorescent protein (GFP), eGFP, CFP, YFP, eYFP, BFP, eBFP, DsRed,and MmGFP. In a specific embodiment, the reporter gene is a lacZ gene.The reporter gene may be any suitable reporter gene.

In one embodiment, the selection cassette comprises a gene selected fromthe group consisting of the following genes: neomycin phosphotransferase(neo^(r)), hygromycin B phosphotransferase (hyg^(r)),puromycin-N-acetyltransferase (puro^(r)), blasticidin S deaminase(bsr^(r)), xanthine/guanine phosphoribosyl transferase (gpt), Herpessimplex virus thymidine kinase (HSV-tk). In a specific embodiment, theselection cassette comprises a neo^(r) gene driven by a UbC promoter.

In one embodiment, the recombinase is selected from the group consistingof the following site-specific recombinases (SSRs): Cre, Flp, and Dre.

In one embodiment, the first and the second RRSs are independentlyselected from a loxp, lox511, lox2272, lox66, lox71, loxM2, lox5171,FRT, FRT11, FRT71, attp, att, FRT, or Dre site.

In one aspect, a method for excising a selection cassette in agenetically modified mouse cell or mouse is provided, comprisingemploying a targeting vector comprising a selection cassette and arecombinase gene operably linked to a 3′-UTR comprising an miRNA asdescribed herein to target a sequence in a donor mouse ES cell, growingthe donor mouse ES cell under selection conditions, introducing thedonor mouse ES cell into a mouse host embryo to form a geneticallymodified embryo comprising the donor ES cell, introducing thegenetically modified embryo into a mouse that is capable of gestatingthe embryo, maintaining the mouse under conditions that allow forgestation, wherein upon differentiation the selection cassette isexcised.

In one aspect, a method is provided for maintaining non-human cells inculture in an undifferentiated state, comprising genetically modifyingan undifferentiated cell with a targeting vector as disclosed hereinthat comprises a selectable marker flanked on each side by site-specificrecombinase recognition sites and a recombinase gene under control of apromoter as disclosed herein and/or comprising a 3′-UTR having an miRNArecognition sequence as described herein, and growing theundifferentiated cell under selective conditions, wherein therecombinase gene is transcribed and the selectable marker is excised inthe event of differentiation of the cell.

In one embodiment, the non-human cell is selected from a pluripotentcell, a totipotent cell, and an induced pluripotent cell. In oneembodiment, the non-human cell is an ES cell. In specific embodiments,the non-human cell is selected from a mouse ES cell and a rat ES cell.

In one aspect, a method is provided for maintaining a culture enrichedwith undifferentiated cells, comprising growing the cells in thepresence of a selection agent, wherein the cells comprise a selectioncassette that allows the cells to grow in the presence of the selectionagent, wherein the selection cassette is flanked 5′ and 3′ by a RSS thatis recognized by a recombinase, wherein the cells comprise a geneencoding the recombinase, wherein the gene encoding the recombinase (a)is operably linked to a promoter selected from the group consisting of aBlimp1 promoter or a Prm1 promoter; or, (b) comprises in its 3′-UTR amiRNA recognition sequence that is a target for an miRNA selected fromthe group consisting of miR-292-3p, miR-290-3p, miR-291a-3p,miR-291b-3p, miR-294, miR-295, miR-302a, miR-302b, miR-302c, miR-302d,miR-367, miR-17, miR-18a, miR-18b, miR-20a, miR-106a, and miR-93; or,(c) is operably linked to a promoter as in (a) and also comprises anmiRNA recognition sequence as in (b).

In one aspect, a cell is provided that comprises a recombinase gene thatis (a) operably linked to a promoter that is inactive or substantiallyinactive in non-germ cells but active in germ cells, and/or (b) operablylinked to a miRNA recognition sequence as described herein; wherein thecell comprises a selection cassette flanked upstream and downstream withRRSs recognized by the recombinase and that are oriented to direct adeletion. In one embodiment, the cell is selected from an inducedpluripotent cell, a pluripotent cell, and a totipotent cell. In oneembodiment, the cell is a mouse cell. In a specific embodiment, themouse cell is a mouse ES cell.

In one embodiment, the germ cell is a sperm lineage cell. In oneembodiment, the promoter that is inactive or substantially inactive innon-germ cells but active in a germ cell is a Prm1 promoter.

In one aspect, a kit is provided, comprising a nucleic acid constructthat comprises a recombinase gene operably linked to a miRNA recognitionsequence as described herein, and a selection cassette flanked 5′ and 3′by RSSs that are recognized by a recombinase expressed by therecombinase gene.

In one aspect, a kit is provided, comprising a nucleic acid constructthat comprises a recombinase gene operably linked to a promoter that isdoes not drive transcription of the recombinase in undifferentiatedcells but that drives transcription of the recombinase in differentiatedcells, and a selection cassette flanked 5′ and 3′ by RSSs that arerecognized by a recombinase expressed from the recombinase gene.

Other embodiments will become apparent from a review of the ensuingdetailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a targeting vector according to an embodiment of theinvention that comprises an miRNA recognition site in the 3′-UTR of arecombinase gene.

FIG. 2 illustrates alignments of miRNAs of the miR-290 cluster andrelated miRNAs, including those abundant in ES cells. SEQ ID NOs are:SEQ ID NO:23 (292-5p); SEQ ID NO:46 (290-5p); SEQ ID NO:21 (291a-5p);SEQ ID NO:47 (291b-5p); SEQ ID NO:48 (293*); SEQ ID NO:49 (294*); SEQ IDNO:50 (295*); SEQ ID NO:51 (302a*); SEQ ID NO:52 (302b*); SEQ ID NO:53(302c*); SEQ ID NO:54 (17*); SEQ ID NO:55 (18*); SEQ ID NO:56 (20a*);SEQ ID NO:26 (292-3p); SEQ ID NO:22 (290-3p); SEQ ID NO:24 (291a-3p);SEQ ID NO:25 (291b-3p); SEQ ID NO:27 (293); SEQ ID NO:28 (294); SEQ IDNO:29 (295); SEQ ID NO:30 (302a); SEQ ID NO:31 (302b); SEQ ID NO:32(302c); SEQ ID NO:33 (302d); SEQ ID NO:34 (367); SEQ ID NO:4 (17); SEQID NO:5 (18a); and SEQ ID NO:8 (20a).

FIG. 3 illustrates an miRNA recognition sequence according to anembodiment of the invention, having four tandem copies of an miR-292-3precognition sequence for insertion in a 3′-UTR of an NL-Crei gene in atargeting vector.

FIG. 4 is a schematic of constructs. Panel A shows a neomycin resistancegene flanked by recombinase recognition sites (RRSs), on a constructhaving a LacZ gene; Panel B shows a human Ub promoter driving expressionof Cre from an NL-Crei gene, on a construct having a hygromycinresistance gene; Panel C shows the construct of Panel B, additionallyincluding a miR recognition sequence 3′ with respect to the NL-Creigene; although not shown, the miR recognition sequence can be present inmultiple copies.

FIG. 5 illustrates a targeting vector of an embodiment of the inventionthat comprises a recombinase gene operably linked to a promoter that isinactive or substantially inactive in undifferentiated (e.g., ES) cells,but is active in differentiated cells.

FIG. 6 shows cell count results for mouse ES cells bearing differentcombinations of constructs of FIG. 4, Panels A, B and C, under differentselection conditions.

FIG. 7 is a schematic of constructs. Panel A shows a neomycin resistancegene flanked by recombinase recognition sites (RRSs), on a constructhaving a LacZ gene; Panel B shows a construct having a GFP gene inreverse orientation flanked by incompatible recombinase recognitionsites (RRSs), wherein GFP is not expressed, and thenrecombinase-mediated inversion to place the GFP in orientation fortranscription.

DETAILED DESCRIPTION

The invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. Theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, particular methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference in their entirety.

Methods and compositions are provided for modifying or removing nucleicacid sequences in a differentiation-dependent manner. The methods andcompositions include promoters or regulatory elements that inducemodification (e.g., inversion) or removal (e.g., excision) of a nucleicacid sequence only when a cell undergoes differentiation or begins adifferentiation process. The methods and compositions also include thosethat employ sequences recognized by miRNAs that are produced and/orfunction in undifferentiated cells but cease to be produced or cease tofunction in differentiated cells. They also include promoters that drivetranscription effectively in differentiated cells, but not effectivelyin undifferentiated cells.

Differentiation-Dependent Regulation of Expression: Promoters and RNAs

An ideal solution to the problem of selectable marker removal fromgenetically modified animals (e.g., knockout mice) would retain theselection cassette in ES cells to enable selection of clones that haveincorporated the targeting vector but promote automatic excision (ormodification, e.g., inversion) of the cassette with essentially 100%efficiency in all cells and tissues of the developing embryo and mousewithout the need for additional treatments or manipulations of targetedES cells or for breeding of mice. Such an ideal solution depends uponthe recombinase that recognizes the recombination sites flanking theselection cassette being inactive, or substantially inactive, inundifferentiated ES cells and then becoming active once the ES cells areincorporated into a developing embryo and begin to differentiate.

One way of achieving differentiation-dependent regulation of therecombinase is to drive the transcription of recombinase mRNA with apromoter that is off in ES cells but comes on once the ES cells begin todifferentiate (e.g., into the cell and tissue types of a developingembryo) or, e.g., that is on in a germ cell such that progeny thatdevelop from the germ cell have expressed the recombinase at a veryearly stage in development. In this way, a selection cassette flanked oneach side by recombinase recognition sites is excised only upondifferentiation (or development). For complete excision of the selectioncassette, the promoter driving recombinase expression would, ideally,remain active in all the cells and tissues of the embryo and mouse.However, certain promoters, e.g., those active in germ cells, might alsobe useful because if the promoter is active in a germ cell of an F0animal, breeding that animal will result in excision of the cassette inall cells and tissues of that animal's progeny.

Embodiments are provided for promoters that are inactive in ES cellsthat have not undergone differentiation, but that are active eitherduring differentiation or when the ES cells begin to differentiate (or,e.g., in germ cells or in germ lineage cells, e.g., in sperm lineagecells). A recombinase gene operably linked to such a promoter will betranscribed, or substantially transcribed, when an ES cell begins todifferentiate (or, e.g., when a cell differentiates into a germ lineagecell, e.g., a sperm lineage cell). If a selection cassette is flanked byrecombinase recognition sites that direct a deletion, then expression ofthe recombinase will cause the differentiating cell to lose theselection cassette and, if the cells are maintained under selectiveconditions, the cells will not survive selection. This affords methodsand compositions for maintaining only undifferentiated ES cells inculture, for maintaining an ES cell culture enriched with respect toundifferentiated cells, and for automatic excision of a selectioncassette upon differentiation of the ES cells while, e.g., the ES cellsare differentiating as donor cells in a host embryo.

In various embodiments, a suitable promoter is selected from a Prm1,Blimp1, Gata6, Gata4, Igf2, Lhx2, Lhx5, Pax3. In a specific embodiment,the promoter is the Gata6 or Gata4 promoter. In another specificembodiment, the promoter is a Prm1 promoter. In another specificembodiment, the promoter is a Blimp1 promoter or fragment thereof, e.g.,a 1 kb or 2 kb fragment of a Blimp1 promoter. A suitable Prm1 promoteris shown in SEQ ID NO:1; a suitable Blimp1 promoter is shown in SEQ IDNO:2 (1 kb promoter) or SEQ ID NO:3 (2 kb promoter).

Differentiation-Dependent Regulation: miRNA Recognition Sequences

Another way of achieving differentiation-dependent regulation of therecombinase is to regulate recombinase expression post-transcriptionallyby miRNA-mediated mechanisms. Micro RNAs (miRNAs) are small RNAs(approximately 22 nucleotides, nt, in length) that associate withArgonaute proteins and regulate mRNA expression by binding to miRNArecognition sites in the 3′-untranslated region (3′-UTR) of mRNA andpromoting inhibition of protein synthesis and destruction of the mRNA(see, e.g., Filipowicz et al. (2008) Mechanisms of post-transcriptionalregulation by microRNAs: are the answers in sight? Nature ReviewsGenetics 9:102-114).

An miRNA interacts with its natural recognition site by forming aWatson-Crick (W-C) base-paired helix between the miRNA's so-called seedsequence—nucleotides 2 through 8 numbering from the 5′ end—and acomplementary sequence in the target mRNA's 3′-UTR. The remainder of themiRNA forms an imperfect helix with the target. This type of imperfectlypaired complex between the target mRNA and the miRNA bound to anArgonaute protein and other components of the RNA-induced silencingcomplex (RISC) triggers the events that result in the inhibition oftranslation of the target mRNA into protein. Another class of naturalsmall RNA known as small interfering RNA (siRNA) is produced by cleavageof long double-stranded RNAs (dsRNAs) into short dsRNAs whose 21 nt (themost frequent length) single strands form a perfect W-C helix over their5′-terminal 19 nucleotides with the last two 3′-terminal nucleotidesleft as unpaired overhangs on each end of the helix. Usually, one strandof a double-stranded siRNA gets loaded into an Argonaute-RISC in amanner similar to miRNAs, but unlike miRNAs, siRNA-loaded RISCs formperfect W-C helices with their target mRNAs and promote cleavage ratherthan translational inhibition. An mRNA cleaved by an siRNA-RISC isusually rapidly degraded by cellular ribonucleases, which usuallyresults in a more severe reduction of the target mRNA and its encodedprotein than that induced by a miRNA-RISC. Researchers have takenadvantage of this difference to regulate expression of genes exogenouslyadded to cells or animals. See, e.g., Mansfield et al. (2004)MicroRNA-responsive ‘sensor’ transgenes uncover Hox-like and otherdevelopmentally regulated patterns of vertebrate microRNA expression,Nature Genetics 36:1079-1083; Brown et al. (2007) Endogenous microRNAcan be broadly exploited to regulate transgene expression according totissue, lineage and differentiation state, Nature Biotech. 25:1457-1467;Brown et al. (2009) Exploiting and antagonizing microRNA regulation fortherapeutic and experimental applications, Nature Reviews Genetics10:578-585.

All miRNAs mentioned refer to mouse miRNAs, i.e., mmu-miRs.

Differentiation-Dependent miRNA Regulation of an Excising Protein

Differential expression of endogenous miRNAs can be advantageously usedto control expression of exogenously added genes in cells and innon-human animals. As discussed above, miRNAs can be potent inhibitorsof translation. Where an miRNA has an expression profile that results ininhibition of its target under one set of conditions, but not underanother, the difference in expression can be exploited to express a geneunder one but not the other set of conditions. Thus, if an endogenousmiRNA can be found that is expressed in undifferentiated cells but notin differentiated cells, the expression of a gene controlled by thatendogenous miRNA can be modulated by placing a recognition sequence (ortarget sequence) for the endogenous miRNA in the gene. miRNA expressionis expected to modulate expression of the target gene even where thetarget gene is an exogenous (or foreign) gene so long as the exogenousgene contains, or is operably linked to, an appropriate miRNArecognition sequence. In this way foreign genes, such as thoseintroduced into a cell or a non-human animal by a targeting vector, canbe placed under the control of an endogenous miRNA. miRNAs that areexpressed only at a certain period in development can be used to silenceexogenous genes during that developmental period. Thus, an miRNA that isexpressed only in undifferentiated cells but not in differentiated cellscan be exploited to silence expression of an exogenous gene in anundifferentiated cell but not following the cell's differentiation, byplacing a recognition sequence recognized by the miRNA in operablelinkage, e.g., in a 3′-UTR, of the exogenous gene to be silenced.

One advantageous application of placing an miRNA recognition sequence ina 3′-UTR that is a target of a developmentally-regulated miRNA is thatnucleic acid sequences in a cell or non-human animal of interest can bemodified or excised by a site-specific recombinase in adevelopmentally-dependent manner. In this application, the sequencedesired to be modified or excised is flanked on each side by RRSs, and arecombinase gene is employed that has a 3′-UTR having a target sequencefor an miRNA that is expressed in a developmentally-dependent manner.Modification or excision may occur by the option of how the RRSs areoriented. The miRNA recognition sequence is selected by determining atwhich developmental stage the recombinase gene is to be activated, andselecting the recognition sequence to bind an endogenous miRNA that isexpressed at the selected developmental stage. For cases of selectioncassette excision discussed herein concerning ES cells, miRNArecognition sequence selection is based on miRNAs that are expressed inundifferentiated cells, but are not expressed in differentiated cells.

Thus, the 3′-UTR of an mRNA of a recombinase is selected so that itcontains one or more (e.g., one to four) miRNA recognition sites thatcomprise perfect (or, in some embodiments, near-perfect) Watson-Crickcomplements of endogenous natural miRNAs such that use of the sequencein the 3′-UTR of the recombinase produces an siRNA-like RNA interference(RNAi) that results in the reduction of both the targeted recombinasemRNA and its encoded recombinase in cells that express the cognatemiRNA.

In various embodiments, the miRNA recognition sites comprise perfect ornear-perfect Watson-Crick complements of endogenous natural miRNA seedsequences, or sufficiently recognize natural miRNA seed sequences suchthat the natural miRNA can bind the target and thus promote inhibitionof expression of the gene bearing the target. In various embodiments,the miRNA recognition sequences are present in one, two, three, four,five, or six or more tandem copies in the 3′-UTR. In variousembodiments, the miRNA recognition sequences are specific for a singlemiRNA, in other embodiments, the miRNA recognition sequences bind two ormore miRNAs. In various embodiments, the miRNA recognition sequences areidentical and designed to bind two or more members of the same miRNAfamily, e.g., the miRNA recognition sequence is a consensus sequence oftwo or more miRNA target sequences. In various embodiments, the miRNArecognition sequences are two or more different recognition sequencesthat bind miRNAs in the same family (e.g., the miR 292-3p family).

miRNAs that are expressed in undifferentiated cells but not indifferentiated cells fall into different miRNA families, or clusters.miRNAs that are abundant in ES cells include, e.g., clusters 290-295,17-92, chr2, chr12, 21, and 15b16. See, e.g., Calabrese et al. (2007)RNA sequence analysis defines Dicer's role in mouse embryonic stemcells, Proc. Natl. Acad. Sci. USA 104(46):18097-18102; Houbaviy et al.(2003) Developmental Cell 5:351-358, and Landgraf et al. (2007) Cell129:1401-1414. Quantification of miRNA in mouse ES cells by sequencingof small RNAs revealed that the ten most abundant miRNAs aremiR-291a-3p, miR-294, miR-292-5p, miR 295, miR-290, miR 293, miR-292-3p,miR-291a-5p, miR-130a, and miR-96. See, Marson et al. (2008) Cell134:521-533, Supplemental FIG. 9. By at least one report based on miRNAquantification by small RNA sequencing, the miR-290-295 clusters miRNAsconstitute about 70% of transcribed miRNAs in ES cells. See, Marson etal. (2008), cited above.

As illustrated herein, the ten most abundant miRNAs present in twospecific mouse ES cell lines was also determined. Mouse ES cell lineVGB6 was isolated at Regeneron Pharmaceuticals, Inc. from a C57BL/6NTacmouse strain (Taconic). Mouse ES cell line VGF1, also isolated atRegeneron Pharmaceuticals, Inc., was isolated from a hybrid 129/B6 F1mouse strain. The ten most abundant miRNAs were identified by microarrayanalysis and found to be miR-292-3p, miR-295, miR-294, miR-291a-3p,miR293, miR-720, miR-1224, miR-19b, miR92a, and miR-130a. The top 20most abundant miRNAs also included, from 11^(th) to 20^(th) mostabundant, miR-20b, miR-96, miR-20a, miR-21, miR-142-3p, miR-709,miR-466e-3p, and miR-183.

For the case of VGB6 cells, quantitative PCR revealed that the 20 mostabundant miRNAs in those cells are, in order, miR-296-3p, miR-434-5p,miR-494, miR-718, miR-181c, miR-709, miR-699, miR-690, miR-1224,miR-720, miR-370, miR-294, miR-135a*, miR-1900, miR-295, miR-293,miR-706, miR-212, and miR-712.

FIG. 2 shows an alignment of miR290 cluster and related miRNAs. The toppanel of FIG. 2 shows miRNAs similar to miR-292-5p (numbered, for thepurposes of the alignment, 1-25), whereas the bottom panel shows miRNAssimilar to miR-292-3p. Boxed areas indicate nucleotide identity. Basedon the sequence similarity shown in the alignments and the functionalresults described herein, a 3′-UTR of a recombinase gene can contain anmiRNA recognition sequence complementary to a miRNA sequence drawn fromthe miR-292-3p family and related miRNAs shown. The miRNA recognitionsequence of the 3′-UTR, in one embodiment, binds an miR-292-3p familymember. The miRNA recognition sequence of the 3′-UTR, in one embodiment,binds an miR-292-3p family member that comprises an identicalWatson-Crick match in its seed sequence to the miRNA recognitionsequence. In another embodiment, the miRNA recognition sequence binds anmiR-292-3p family member and has about 85%, about 90%, about 95%, 96%,97%, 98%, or 99% identity to a sequence of FIG. 2.

The alignment of FIG. 2 showing similarity among 292-3p family membersreveals a near-identical seed sequence of 5′-AAGUGCC-3′ located at bases2-8 from the 5′ end of the miRNAs of the 292-3p family. This presumablyhelps members of the 292-3p family bind mRNAs that contain theWatson-Crick complement of 5′-AAGUGCC-3′ in their 3′-UTRs. The remainderof the miRNA molecule can form base pairs with the target, butcomplementarity is not typically perfect for animal miRNAs and theirtargets.

In one embodiment, the miRNA recognition sequence operably linked to therecombinase gene comprises a seed sequence that comprises a sequencethat is identical to 5′-AAGUGCC-3′. In one embodiment, the miRNArecognition sequence operably linked to the recombinase gene comprises aseed sequence that is identical to 5′-AAGUGCC-3′ except for a singlenucleic acid substitution. In a specific embodiment, the secondnucleotide of the seed sequence is a G or an A. In a specificembodiment, the third nucleotide of the seed sequence is a G or a U. Ina specific embodiment, the final position of the seed sequence is a C.In a specific embodiment, the final position of the seed sequence is aU. In a specific embodiment, the final position of the seed sequence isan A.

In one embodiment, the miRNA recognition sequence operably linked to therecombinase gene comprises a seed sequence that is perfectlycomplementary to a seed sequence of an miRNA expressed in an ES cell butnot expressed in a differentiated cell, the miRNA is one of the ten mostabundant miRNAs expressed in the ES cell in an undifferentiated state,and the miRNA recognition sequence further comprises 14-18 furthernucleotides that are about 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to an miRNA naturally expressed in the undifferentiated EScell, and wherein the presence of the miRNA recognition sequence in the3′-UTR of the recombinase gene results in a decrease of expression of atleast 50% as compared with a recombinase gene with a 3′-UTR that lacksthe miRNA recognition sequence. In a specific embodiment, the decreasein expression of the recombinase is at least 60%, at least 70%, at least80%, at least 90%, or at least 95%.

In one embodiment, the miRNA recognition sequence comprises a seedsequence of an miRNA selected from miR-292-3p and miR-294. In a specificembodiment, the miRNA recognition sequence further comprises a non-seedsequence that is at least 90% identical with a non-seed sequence of anmiRNA selected from the group consisting of miR-292-3p and miR-294. In aspecific embodiment, the miRNA recognition sequence further comprises anon-seed sequence that is at least 95% identical with a non-seedsequence of an miRNA selected from the group consisting of miR-292-3pand miR-294.

In one embodiment, the miRNA recognition sequence operably linked to therecombinase gene is recognized by an miRNA selected from miR-292-3p,miR-290-3p, miR-291a-3p, miR-291b-3p, miR-294, miR-295, miR-302a,miR-302b, miR-302c, miR-302d, miR-367, miR-17, miR-18a, miR-18b,miR-20a, miR-106a, and miR-93.

In one embodiment, the miRNA recognition sequence binds miR-292-3p,miR-290-3p, miR-291a-3p, miR-291b-3p, miR-294, miR-295, miR-302a,miR-302b, miR-302c, miR-302d, miR-367, miR-17, miR-18a, miR-18b,miR-20a, miR-106a, or miR-93, and is one of the 20 most abundant miRsspecifically expressed in the target cell. In one embodiment, the miRNAis one of the 10 most abundant miRNAs expressed in the target cell. Inone embodiment, the miRNA is one of the five most abundant miRNAsexpressed in the target cell. In one embodiment, the target cell is amouse ES cell and the miRNA is selected from an miR of Table 2. In oneembodiment, the miR is selected from the group consisting of miR-292-3p,miR-290-3p, miR-291a-3p, miR-291b-3p, miR-294, miR-295, miR-302a,miR-302b, miR-302c, miR-302d, miR-367, miR-17, miR-18a, miR-18b,miR-20a, miR-106a, or miR-93, and a combination thereof. In oneembodiment, the miRNA recognition sequence comprises a sequence that iscomplementary to a seed sequence of one of miR-292-3p, miR-290-3p,miR-291a-3p, miR-291 b-3p, miR-294, miR-295, miR-302a, miR-302b,miR-302c, miR-302d, miR-367, miR-17, miR-18a, miR-18b, miR-20a,miR-106a, or miR-93, and the remainder of the miRNA recognition sitecomprises a non-seed sequence that is about 85%, 90%, 95%, 96%, 97%,98%, or 99% complementary to a non-seed sequence independently selectedfrom one of miR-292-3p, miR-290-3p, miR-291a-3p, miR-291b-3p, miR-294,miR-295, miR-302a, miR-302b, miR-302c, miR-302d, miR-367, miR-17,miR-18a, miR-18b, miR-20a, miR-106a, or miR-93.

In one embodiment, the miRNA recognition sequence contains a sequencethat is a perfect Watson-Crick match to a seed sequence of an miRNA ofTable 2, and the remainder of the miRNA recognition sequence (outside ofthe sequence that perfectly matches the miRNA seed sequence) is 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to the non-seed sequence of anmiRNA of Table 2. In one embodiment, the miRNA is selected from thegroup consisting of miR-292-3p, miR-295, miR-294, miR-291a-3p, miR-293,miR-720, miR-1224, and a combination thereof. Sequences of miRNAs areprovided in Table 1 below.

TABLE 1 mmu-miRNA Sequences SEQ miR Sequence ID NO   17CAAAGUGCUUACAGUGCAGGUAG  4   18a UAAGGUGCAUCUAGUGCAGAUA  5   18bUAAGGUGCAUCUAGUGCUGUUAG  6   19b UGUGCAAAUCCAUGCAAAACUGA  7   20aUAAAGUGCUUAUAGUGCAGGUAG  8   20b CAAAGUGCUCAUAGUGCAGGUAG  9   21UAGCUUAUCAGACUGAUGUUGA 10   92a UAUUGCACUUGUCCCGGCCUG 11   93CAAAGUGCUGUUCGUGCAGGUAG 12   96 UUUGGCACUAGCACAUUUUUGCU 13  106aCAAAGUGCUAACAGUGCAGGUAG 14  130a CAGUGCAAUGUUAAAAGGGCAU 15  135a*UAUAGGGAUUGGAGCCGUGGCG 16  142-3p UGUAGUGUUUCCUACUUUAUGGA 17  181cAACAUUCAACCUGUCGGUGAGU 18  183 GUGAAUUACCGAAGGGCCAUAA 19  212UAACAGUCUCCAGUCACGGCCA 20  291a-5p CAUCAAAGUGGAGGCCCUCUCU 21  290-3pAAAGUGCCGCCUAGUUUUAAGCCC 22  292-5p ACUCAAACUGGGGGCUCUUUUG 23  291a-3pAAAGUGCUUCCACUUUGUGUGC 24  291b-3p AAAGUGCAUCCAUUUUGUUUGU 25  292-3pAAAGUGCCGCCAGGUUUUGAGUGU 26  293 AGUGCCGCAGAGUUUGUAGUGU 27  294AAAGUGCUUCCCUUUUGUGUGU 28  295 AAAGUGCUACUACUUUUGAGUCU 29  302aUAAGUGCUUCCAUGUUUUGGUGA 30  302b UAAGUGCUUCCAUGUUUUAGUAG 31  302cAAGUGCUUCCAUGUUUCAGUGG 32  302d UAAGUGCUUCCAUGUUUGAGUGU 33  367AAUUGCACUUUAGCAAUGGUGA 34  370 GCCUGCUGGGGUGGAACCUGGU 35  434-5pGCUCGACUCAUGGUUUGAACCA 36  494 UGAAACAUACACGGGAAACCUC 37  690AAAGGCUAGGCUCACAACCAAA 38  706 AGAGAAACCCUGUCUCAAAAAA 39  709GGAGGCAGAGGCAGGAGGA 40  712 CUCCUUCACCCGGGCGGUACC 41  718CUUCCGCCCGGCCGGGUGUCG 42  720 AUCUCGCUGGGGCCUCCA 43 1224GUGAGGACUGGGGAGGUGGAG 44 1900 GGCCGCCCUCUCUGGUCCUUCA 45

Differentiation-Dependent Excision of Selection Cassettes

To create various embodiments of a self-deleting selection cassettewhose excision is regulated by miRNA control of recombinase geneexpression, a standard selection cassette is modified by insertion of arecombinase gene unit that comprises a promoter, which may or may not beactive in ES cells but is active in embryonic stages after theblastocyst, linked to the protein coding sequence of a site-specificrecombinase, e.g., Cre, Flp, or Dre, followed by a sequence encoding the3′-UTR of the recombinase mRNA, into which is inserted a copy of, ormultiple copies of, a sequence complementary to one or more miRNAs thatare expressed in ES cells but not in any of the cells of the developingembryo or mouse, and terminated with a polyadenylation signal. Themodified selection cassette with the inserted miRNA-regulatablerecombinase gene unit is flanked by recognition sites for therecombinase whose gene has been inserted. The orientation of theflanking recombinase recognition sites is such that the recombinase willcatalyze the deletion of the modified selection cassette, including therecombinase gene. Embodiments are also possible where the selectioncassette is on a separate construct, in which case the recombinase worksin trans.

In one embodiment, the recombinase gene is a Cre recombinase gene. Inone embodiment, the Cre recombinase gene further comprises a nuclearlocalization signal to facilitate localization of Cre to the nucleus(e.g., the gene is an NL-Cre gene). In one embodiment, the Crerecombinase gene comprises an intron (e.g., the gene is a Crei gene),such that the Cre recombinase is not functional in bacteria. In aspecific embodiment, the Cre recombinase gene further comprises anuclear localization signal and an intron (e.g., NL-Crei).

An example of part of a targeting vector designed to create a knockoutallele in which the selectable marker is included within aDifferentiation-Dependent Self-Deleting Cassette, or DDSDC, isillustrated in FIG. 1. The rectangle indicates the portion of thetargeting vector that inserts at the targeted locus. The thick blacklines flanking the rectangle represent parts of the mouse DNA homologyarms that promote homologous recombination at the targeted locus. In theexample shown, a reporter gene cassette (a common feature of knockoutalleles) is shown in which the coding sequence of a reporter protein,such as β-galactosidase or green fluorescent protein, is fused to thetargeted gene in such a way as to report the transcriptional activity ofthe target gene's promoter. The region between the solid triangles(i.e., between the recombinase recognition sites) represents an exampleof a Differentiation-Dependent Self-Deleting Cassette: the left portionis the selection cassette consisting of gene that encodes a protein thatimparts drug resistance (drug^(r)), such as neomycin phosphotransferase,which imparts resistance to the drug G418; the right portion is a genethat encodes a site-specific recombinase, e.g., Cre, Flp, or Dre,containing in its 3′-UTR multiple target sites for one or more EScell-specific miRNAs. The DDSDC is flanked by the sites (blacktriangles) recognized by the encoded recombinase, for example, loxP sitefor the Cre recombinase, FRT sites for the Flp recombinase, or rox sitesfor the Dre recombinase, oriented such that recombinase action at thesites will promote excision of the DDSDC. The promoters drivingexpression of the drug^(r) and recombinase genes are indicated by “pro”with bent arrows above denoting the direction of transcription. In theexample shown the drug^(r) and recombinase genes are oriented in thesame transcriptional direction, but they could be oriented in eitherdirection. Polyadenylation signals are indicated by “p(A).”

When a modified selection cassette containing the miRNA-regulatablerecombinase gene is incorporated into a targeting vector and introducedinto mouse ES cells by standard methods of gene targeting known in theart, expression in the ES cells of miRNAs that recognize their targetsequence in the 3′-UTR of the recombinase mRNA transcribed from theselection cassette will promote a reduction in recombinase proteinsynthesis to levels that are too low to substantially excise theselection cassette and, therefore, will permit selection ofdrug-resistant colonies. As long as the targeted ES cells remainundifferentiated, their endogenous ES-cell-specific miRNAs will controlexpression of the recombinase and permit drug selection of ES cells thatcontain the targeted construct. Targeted clones that differentiate awayfrom the ES cell state, however, will lose expression of the EScell-specific miRNAs, relieving inhibition of recombinase expression,which will result in substantial excision of the selection cassette andloss of drug resistance. Therefore, differentiated clones will be killed(i.e., not survive selection) and would not be used to generategene-modified mice. Undifferentiated, drug-resistant gene-targetedclones, upon injection into an early mouse embryo (e.g., a premorula,e.g., 8-cell stage embryo, or a blastocyst) will become integrated intothe inner cell mass that will ultimately contribute to the developingmouse embryo.

When the injected embryos are transplanted into a surrogate mother andbegin to differentiate along a normal developmental path, expression ofES cell-specific miRNAs will wane and the recombinase will be expressedand become active wherever the recombinase gene is transcribed. Drivingrecombinase expression with a ubiquitously active promoter (e.g., aphosphoglycerate kinase, β-actin, ubiquitin promoter, or other promoter)will ensure that the recombinase will have ample opportunity to excisethe selection cassette from all or most cell types during the course ofdevelopment, resulting in pups born devoid of the selection cassette atthe targeted locus. These new-born mice would be ready for phenotypicstudy without concerns about interference by a selection cassette.

In one embodiment, a method for preparing an ES cell culture that lacksviable differentiated cells is provided, comprising introducing into anES cell a selection cassette and a recombinase gene, wherein either theselection cassette alone or the recombinase gene and the selectioncassette are flanked by RRSs recognized by the recombinase, and therecombinase gene is operably linked to an miRNA target sequence asdescribed herein; growing the ES cell to form an ES cell culture,wherein cells that differentiate in culture lose the selection cassetteand expire, thereby forming an ES cell culture that lacks orsubstantially lacks viable differentiated cells, or comprises asubstantially reduced number of viable differentiated cells.

In one embodiment, a method for preparing a population of donor mouse EScells enriched with respect to undifferentiated ES cells is provided,comprising employing an ES cell as described herein that comprises aselection cassette and a recombinase operably linked to a miRNArecognition sequence as described herein, growing the ES cell to form anES cell culture, and employing the ES cell culture as a source of donorES cells for introduction into a mouse host embryo. In one embodiment,the ES cell culture is enriched with respect to undifferentiated EScells by about 10%, 20%, 30%, 40%, or 50% or that more in comparison toa culture in which ES cells do not comprise the miRNA recognitionsequence operably linked to the promoter, and the cells are grown in amedium that requires the selection cassette for survival. In oneembodiment, the ES cell culture comprises no more than one viabledifferentiated cell per 100 cells, no more than one viabledifferentiated cell per 200 cells, per 300 cells, per 400 cells, per 500cells, per 1,000 cells, or per 2,000 cells. In a specific embodiment,the ES cell culture comprises no viable differentiated cells.

In one embodiment, a differentiated mouse cell is provided, comprising arecombinase gene operably linked to a miRNA target sequence as describedherein, and at least one recombinase recognition site. In oneembodiment, the differentiated mouse cell is in a mouse embryo. In oneembodiment, the differentiated mouse cell is in a tissue of a mouse. Inone embodiment, the differentiated mouse cell further comprises agenetic modification selected from a knock-in, a knockout, a mutatednucleic acid sequence, and an ectopically expressed protein.

In one embodiment, a method for making a genetically modified mouse thatlacks a selection cassette is provided, comprising (a) introducing intoa mouse host embryo a donor mouse ES cell that comprises (i) a selectioncassette flanked 5′ and 3′ with RSSs oriented to direct a deletion, anda recombinase gene operably linked to a promoter that is inactive inundifferentiated cells but active in differentiated cells; or, (ii) aselection cassette flanked upstream and downstream with RSSs oriented todirect a deletion, and a recombinase gene operably linked to an miRNAtarget sequence as described herein; (b) introducing the embryo into asuitable host mouse for gestation; and (c) following gestation obtaininga mouse that lacks the selection cassette. In one embodiment, the F0generation mouse lacks the selection cassette. In one embodiment, the F0mouse is a chimera wherein less than all cells of the mouse lack theselection cassette, and upon breeding the F0 mouse an F1 generationmouse is obtained that lacks the selection cassette.

In one embodiment, a method for identifying differentiated cells inculture is provided, comprising introducing into an undifferentiatedcell (a) a marker cassette that contains a detectable marker gene inantisense orientation, wherein the marker cassette is flanked upstreamand downstream with RRSs oriented to direct an inversion; and, (b) arecombinase gene operably linked to (i) a promotor that is inactive inundifferentiated cells but active in differentiated cells, and/or (ii) amiRNA target sequence as described herein; wherein the cell begins todifferentiate and the recombinase is expressed and places the detectablemarker gene in sense orientation, the detectable marker gene istranscribed, and the cell that begins to differentiate is identified bythe expression of the detectable marker. In one embodiment, thedetectable marker is a fluorescent protein, and the cell that begins todifferentiate is identified by detecting fluorescence from the cell.

EXAMPLES

The following examples are provided to describe to those of ordinaryskill in the art a disclosure and description of how to make and useembodiments of the invention, and are not intended to limit the scope ofwhat the inventors regard as their invention. Efforts have been made toensure accuracy with respect to numbers used (e.g., amounts,temperature, etc.) but some experimental errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is average molecular weight, temperature is expressedby degrees Celsius, and pressure is at or near atmospheric.

Example 1 miRNA Abundance in VGB6 and VGF1 ES Cells

Abundance of miRNAs in mouse ES cell lines VGB6 and VGF1 was determinedby microarray analysis. Briefly, small RNAs were purified from the EScells, labeled, and used to probe Agilent miRNA arrays. Abundancereadings from array analysis are expressed as hybridization signalintensities.

The twenty most abundant miRNAs are shown based on triplicate readingsfor VGB6 and for VGF1 in Table 2.

TABLE 2 ES Cell miRNA Microarray Abundance Analysis miRNA Abundance(avg., n = 3) miRNA VGB6 VGF1 miR-292-3p 111769 127534 miR-295 103566117946 miR-294 98411 116437 miR-291a-3p 85478 99872 miR-293 73418 11048miR-720 47419 107611 miR-1224 41173 19402 miR-19b 28868 37820 miR-92a27722 29698 miR-130a 22974 21864 miR-20b 18677 25450 miR-96 16218 12988miR-20a 15654 20744 miR-21 15427 29023 miR-142-3p 10369 7152 miR-70910078 3117 miR-466e-3p 9645 8797 miR-183 8714 7346

The microarray abundance analysis revealed that the top ten abundantmiRNAs (ranked by VGB6 abundance) fell largely within the miRNA-290cluster.

Abundance of miRNAs in VGB6 cells was also determined by quantitativeRT-PCR. The qRT-PCR results showed that miRNA-290 family and themiRNA-17-92 family were among the most abundant miRNAs in VGB6 cells.

Example 2 Targeting Vector with miRNA in a Recombinase 3′-UTR

A targeting vector in accordance with an embodiment of the invention isconstructed by employing, from 5′ to 3′ with respect to transcription ofthe targeted gene, a 5′ homology arm, a lacZ reporter gene followed by apolyA sequence, a loxP site, a neo^(r) gene driven by a UbC promoter, apolyA sequence, a promoter driving expression of a Cre recombinase gene,a 3′-UTR containing four copies of an miR-292-3p target site (see FIG.3), a polyA sequence, a loxP site, and a 3′ homology arm.

Construction of a quadruple miR-292-3p target site by annealing of 4oligos. To assemble a quadruple miR-292-3p target site,oligodeoxynucleotides S1 and AS1 of FIG. 3 are annealed to produce thehybrid S1:AS1 with Nhe I and Mlu I single-stranded overhangs,oligodeoxynucleotides S2 and AS2 are annealed to produce the hybridS2:AS2 with Mlu I and Xma I single-stranded overhangs, S1:AS1 and S2:AS2are annealed through their Mlu I single-stranded overhangs, and theannealed hybrids are inserted into Nhe I and Xma I sites in the 3′-UTRof a recombinase gene. Sequences that are perfect Watson-Crickcomplements of the mouse miR-292-3p microRNA are labeled “miR-292-3ptarget” in FIG. 3. Alternatively, a synthetic piece of DNA carrying fourmiR-292-3p recognition sequences are placed in the 3′-UTR of a Crerecombase.

The targeting vector containing the miRNA target site of FIG. 3 isemployed by homologous recombination of the targeting vector in a mouseES cell, growing the ES cell under conditions that prevent ES celldifferentiating, introducing the ES cell into an early stage embryo(e.g., a premorula) or a blastocyst, and introducing the embryo into asurrogate mother.

Since miR-292-3p is expressed in ES cells, the selection cassette shouldremain in the ES cell genome during growth and selection of ES cellsgenetically modified by the targeting vector. To the extent that one ormore ES cells bearing the targeting vector would differentiate inculture, those cells would lose the selection cassette and not surviveselection.

Once placed into the embryo, the ES cell would divide and populate theembryo. As ES cells within the embryo differentiated, the level ofmiR-292-3p in the differentiating cell would drop substantially or fallto essentially none. As a result, repression of expression of the Crerecombinase would be relieved, the Cre would express, and the floxedcassette would be excised. Consequently, all or substantially all of thetissues of a mouse born from the surrogate mother would lack theselection cassette.

Example 3 Placement of an miRNA in a 3′-UTR of a Reporter Gene

A commercially available luciferase expression vector was modified byadding a single copy of an exact Watson-Crick complement of an miRNAexpressed in ES cells to the 3′-UTR of the luciferase gene. The vectorwas transiently transfected into the ES cells, and luciferase expressionwas knocked down as compared to luciferase expression from a vectorlacking the miRNA target sequence. This experiment established thatplacement of an exogenous miRNA into a 3′-UTR of a reporter gene resultsin an operable unit that can effectively repress gene expression.

Example 4 miRNA Control of Cre Expression in Cells and Mice: Selection

Mouse ES cells from a hybrid line (129S6×C57BL6; F1) were electroporatedwith a first LacZ-containing construct having a floxed neomycinresistance cassette (FIG. 4, Panel A). Cells surviving neomycinselection were then also electroporated with a second constructcontaining a ROSA26-driven hygromycin resistance cassette and ahUbC-driven NL-Crei gene (FIG. 4, Panel B), or the same second constructbut wherein the NL-Crei gene is operably linked to four tandem copies ofan miR 292-3p target sequence placed in the NL-Crei 3′-UTR (FIG. 4,Panel C).

The ES cells were genotyped for the presence of the transfectedconstruct and screened for copy number, then introduced into 8-cellstage Swiss Webster embryos using the VELOCIMOUSE® method (see, U.S.Pat. Nos. 7,659,442, 7,576,259, 7,294,754, and Poueymirou et al. (2006)F0 generation mice fully derived from gene-targeted embryonic stem cellsallowing immediate phenotypic analyses, Nat. Biotech. 25:91-99; eachhereby incorporated by reference). E10.5 embryos fully derived from thetransfected hybrid ES cells were analyzed for the presence of thetransfected cassettes. Results are shown in Table 3 (Cre 1,2=constructwith NL-Crei lacking miRNA in 3′-UTR; Cre-miR 1,2,3=construct withNL-Crei and miR 292-3p target sequence in 3′-UTR). Using theseconstructs and maintaining the ES cells under conditions selected toretain pluripotency and in the presence of hygromycin or G418 andhygromycin, only those cells that contain the floxed neo cassette but donot express Cre will survive G418 selection. Overall, in all studies,46% of ES cell clones carrying a floxed selection cassette and amiR-regulated NL-Crei gene exhibited complete deletion of the selectioncassette either in embryos or in live-born mice.

Genotyping results for the embryos (whole embryo analyzed) and mice (sixtissues analyzed) indicate that regulation of the Cre recombinase by theES cell-specific miRNAs is relieved upon differentiation anddevelopment, as early as day 10.5 of gestation. Live-born mice can beobtained that lack the floxed selection cassette, when multiple tissuesare examined.

TABLE 3 Genotyping of E10.5 Embryos and Mice Total Neo Deleted Total NeoDeleted ES Cell Embryos Embryos Mice Mice Clone Selection (n) (n) (%)(n) (n) (%) Parental — 4 0 0 2 0 0 Cre 1 Hyg 9 9 100 3 3 100 Cre 2 Hyg 44 100 3 3 100 Cre-miR 1 Hyg + neo 6 5 83.3 3 3 100 Cre-miR 2 Hyg + neo 81 12.5 n.d. n.d. n.d. Cre-miR 3 Hyg + neo n.d. n.d. n.d. 1 1 100

Gentoyping results established that ES cells transfected with aconstruct comprising NL-Crei operably linked to four copies of a miR292-3p target sequence (in the NL-Crei gene 3′-UTR) and selected in G418(i.e., selected for the presence of neo expression) yielded embryos thatlacked the neomycin resistance gene (the floxed selection cassette).These results establish that ES donor cells bearing a NL-Crei geneoperably linked to a target miRNA sequence for an miRNA expressed in EScells but not in differentiated cells can be propagated in culture usinga suitable selection cassette and, when introduced into a host embryo,the ES cells can perform an automatic deletion of the cassette when theydifferentiate (and thus no longer express the miRNA that binds to thetarget miRNA sequence). Therefore, ES cells that bear a selection ormarker cassette flanked with recombinase recognition sites, and arecombinase gene operably linked to a miRNA target sequence for a miRNAthat is expressed in ES cells but not in differentiated cells, can bemaintained in culture such that pluripotency is maintained, and afterintroduction of the cells into a host embryo and differentiation, theselection or marker cassette is automatically removed.

In in vitro culture studies, cells bearing the NL-Crei gene but lackingthe miRNA recognition site in the 3′-UTR (FIG. 4, Panel B) grew well inthe presence of hygromycin, but largely expired when G418 was added(FIG. 6, left), indicating that Cre expressed effectively and removedthe floxed neo resistance cassette. Cells bearing the NL-Crei geneoperably linked to four tandem copies of miR 292-3p target sequence inthe NL-Crei 3′-UTR grew well in hygromycin, and also nearly as well inhygromycin and G418 (FIG. 6, right), indicating that the miR recognitionsequence inhibited expression of Cre to a significant extent.Essentially the same results were obtained using two different hybridclones, as well as two clones of an inbred BL/6 ES cell line transfectedwith the same constructs (data not shown).

In separate experiments, similar cells bearing the constructs describedabove were grown in the presence of one of either hygromycin, G418, orboth, in either the presence or absence of LIF, and/or in the presenceor absence of retinoic acid for seven or eight days. Control cells thatbore a floxed neo cassette and a constitutive Cre substantially expiredin the presence of G418, whereas cells in which the NL-Crei gene waslinked to the miR 292-3p target sequences had a substantially lowerdeath rate (as low as about 0-25%, compared with cells lacking the miRtarget sequence; based on colony counts; data not shown). Cells thatbore the NL-Crei gene operably linked to the miR 292-3p target sequencesexhibited about a 2- to 3-fold higher death rate—when grown without LIFand in the presence of retinoic acid, hygromycin, and G418—than controlcells (based on colony counts; data not shown). Similar results were hadwith a similar experiment using C57BL/6 ES cells (VGB6 cells).

These results establish that ectopic miRNA recognition sequences caneffectively inhibit expression of an ectopically expressed recombinaseoperably linked to the miRNA recognition sequences, and that thisphenomenon can be used to control recombination of recombinase-flankedcassettes in ES cells, including for automatic expression or deletion ofthe recombinase-flanked cassettes. The results also establish thatoperably linking an ES cell-specific miRNA recognition sequence to therecombinase gene can assist in maintaining an ES cell culture enrichedwith respect to undifferentiated ES cells by reducing viability ofdifferentiated cells in a selection medium.

Example 5 miRNA Control of Cre Expression in Cells and Mice: Markers

Mouse ES cells were transfected as described above with a firstconstruct containing a GFP gene in antisense orientation flanked bynonidentical recombinase recognition sites (FIG. 7, Panel B) oriented todirect an inversion, and a second construct containing a ROSA26-drivenhygromycin resistance cassette and a hUbC-driven NL-Crei gene (FIG. 4,Panel B), or the same second construct but wherein the NL-Crei gene isoperably linked to four tandem copies of an miR 292-3p target sequenceplaced in the NL-Crei 3′-UTR (FIG. 4, Panel C). Followingelectroporation, cells were grown in the presence of hygromycin andassayed by FACS for GFP expression.

GFP expression analysis of 2×10⁴ cells each for four separate clonesexpressing Cre from a hUbC-driven construct in the absence of an miRNAtarget sequence in the Cre gene 3′-UTR (FIG. 4, Panel B) was conductedon a MoFlo™ (Beckman Coulter) FACS machine. An average of 85.6% of cellsexhibited GFP fluorescence. GFP expression analysis of 2×10⁴ cells eachfor four separate clones bearing four tandem copies of miR 292-3p in the3′-UTR of a NL-Crei gene (FIG. 4, Panel C) an average of 46.5% of thecells exhibited GFP fluorescence. Eight other clones similarly testedwith or without the miR 292-3p in the NL-Crei 3′-UTR yielded similarresults: an average of 91.3% cells expressed GFP in the absence of themiRNA target sequence, whereas an average of only 48.7% of cellsexpressed GFP in the presence of the miR 292-3p target sequence. Neitherculture was inspected for the presence of differentiating cells.

In contrast, clones containing a construct having an NL-Crei gene havingfour tandem copies of a miR 291a-5p target sequence, or four tandemcopies of a miR 1-1 target sequence, in its 3′-UTR showed essentially nodifference in GFP expression as measured by FACS as compared with clonescontaining the same NL-Crei gene but lacking any miR target sequences.These results establish that inhibition of Cre gene expression wasspecific for the miR 292-3p target sequences, and not merely a randommiRNA target sequence.

In another experiment, clones containing a construct having an NL-Creigene with four copies of an miRNA recognition sequence for miR 292-3p,miR 291a-5p, miR 1-1, or miR 294 in its 3′-UTR were tested in a similarFACS assay for GFP expression. Four clones of each were tested. Averagepercent GFP on FACS analysis revealed that neither clones containing themiR 291a-5p recognition sequence nor the miR 1-1 recognition sequenceshowed inhibition of Cre expression (percent GFP greater than or equalto 96%), whereas an average of only about 46.5% of all cells containingmiR 292-3p recognition sequence, and an average of only about 37.0% ofall cells containing the miR 294 recognition sequence, exhibited GFPexpression.

None of the cells were selected for maintenance of pluripotency in thecourse of this experiment. This experiment establishes that recombinaseactivity can effectively be reduced by operably linking the recombinasegene to a miRNA target sequence in the 3′-UTR of the recombinase gene.These results also establish that it is possible to select for ES cells,from a mixture of cells (using FACS) that have not differentiated, e.g.,that have not ceased expressing miRNAs expressed only in ES cells, orseparating out cells that have ceased to express miRNAs expressed onlyin ES cells.

Example 6 Promoter Control of Expression: Prm1 and Blimp1

Mouse ES cells were transfected as described above with a firstconstruct containing a GFP gene in reverse orientation flanked byrecombinase recognition sites directing an inversion (FIG. 7, Panel B),and a second construct containing a NL-Crei gene driven by either a Prm1promoter, a Blimp1 (1 kb fragment), or a Blimp1 (2 kb fragment) promoter(FIG. 5). Following electroporation, cells were grown in the presence ofhygromycin and assayed by FACS for GFP expression. The ES cells weregrown under conditions sufficient to maintain pluripotency.

Four clones having a Prm1 promoter driving Cre expression, four cloneshaving a Blimp1 (1 kb fragment) driving Cre expression, and four cloneshaving a Blimp1 (2 kb fragmetn) driving Cre expression were analyzed byphase contrast microscopy and by fluorescence microscopy to detectGFP-expressing cells. Cell counts were averaged and less than 1% ofcells having the Prm1 promoter were GFP-positive, less than 0.1% ofcells having the Blimp1 (1 kb fragment) promoter were GFP-positive, andless than 0.1% of cells having the Blimp1 (2 kb fragment) promoter wereGFP-positive. These results establish that the Prm1 promoter and bothBlimp1 promoter fragments were inactive in ES cells grown underconditions sufficient to support pluripotency. Thus, these promoters canbe operably linked to a recombinase in ES cells maintained underpluripotency conditions, without any significant expression of therecombinase. Upon loss of pluripotency or differentiation, or uponactivation in a germ cell, the promoters are expected to effectivelydrive Cre expression.

FACS analysis of ES cell clones comprising a Prm1-driven NL-Crei gene, a1 kb Blimp1-driven NL-Crei gene, and a 2 kb Blimp1-driven NL-Crei genesupported the microscopy results described above. Essentially noGFP-expressing cells were detected in non-differentiated ES cell samples(data not shown).

One clone bearing the Blimp1 (2 kb fragment) was used as a donor ES cellto generate a mouse using the VELOCIMOUSE® method as described above,with a Swiss Webster host embryo. E13.5 F0 generation embryos wereharvested and examined for donor and host contribution. They appearednormal and genotyping results (donor cell vs. host embryo contribution)established that five embryos were essentially fully ES cell-derived(i.e., derived from the donor ES cell bearing a Blimp1 (2 kbfragment)-driven NL-Crei gene and the reverse-oriented GFP construct).Fluorescence analysis of one of the five embryos revealed a significantand apparently homogenous widespread fluorescence over background, wherebackground was fluorescence in embryos derived wholly from host cells(i.e., embryos lacking a GFP gene). These results establish that, upondifferentiation, the donor ES cells effectively drive transcription ofthe NL-Crei gene from the Blimp1 promoter, which produces Cre and placesthe inverted GFP gene in orientation for transcription, and GFP iseffectively transcribed.

Consistent with the GFP fluorescence seen in embryos, genotyping of atail biopsy from live-born mice of the same genotype as the embryosdescribed above (with NL-Crei operably linked to a Blimp1 promoter)revealed that the embryos were mosaic with respect to the Cre-mediatedrearrangement of the GFP allele; both rearranged and unrearrangedalleles were detected in tail DNA of live-born mice. Blimp1 is known todrive expression in some lineages, but not others. Blimp1 is alsowell-known to be active in cells of male gametogenic lineage (leading tosperm). Thus, it is expected that breeding F0 mice will result in an F1generation that exhibits uniform expression of GFP in all cells andtissues.

Genotyping of a tail biopsy from live-born mice of the same genotype asthe embryos described above (with NL-Crei operably linked to a Prm1promoter) revealed no detectable Cre-driven rearrangement of the GFPallele, as expected. The Prm1 promoter is expected to drive expressionin sperm lineage cells. Thus, it is expected that breeding F0 mice willresult in an F1 generation that exhibits uniform expression of GFP inall cells and tissues.

1-20. (canceled)
 21. A mouse embryonic stem (ES) cell, comprising: (i) aselection cassette comprising a selectable marker operably linked to afirst promoter, wherein the selection cassette is flanked upstream anddownstream by first and second recombinase recognition sites orientatedto direct an excision; and (ii) a recombinase cassette comprising asecond promoter operably linked to a gene encoding a recombinase thatrecognizes the first and second recombinase recognition sites, whereinthe recombinase gene is separate from the selection cassette and isoperably linked to a 3′-UTR that comprises one or more miRNA recognitionsites that are recognized by one or more miRNAs that are active in anundifferentiated cell but are not active in a differentiated cell sothat the recombinase gene is expressed in the differentiated cell andworks in trans to excise the nucleotide sequence flanked by the firstand second recombinase recognition sites from the genome of thedifferentiated cell.
 22. The mouse ES cell of claim 21, furthercomprising a reporter gene.
 23. The mouse ES cell of claim 22, whereinthe reporter gene is operably linked to the promoter of a targeted genein the mouse ES cell.
 24. The mouse ES cell of claim 22, wherein thereporter gene is selected from the group consisting of a luciferasegene, a LacZ gene, a GFP gene, an eGFP gene, a CFP gene, a YFP gene, aneYFP gene, a BFP gene, an eBFP gene, a DsRed gene, and a MmGFP gene. 25.The mouse ES cell of claim 21, wherein the gene encoding the recombinaseis flanked by third and fourth recombinase recognition sites recognizedby the recombinase.
 26. The mouse ES cell of claim 21, wherein theselection cassette comprises a gene that encodes neomycinphosphotransferase (neo^(r)), hygromycin B phosphotransferase (hyg^(r)),puromycin-N-acetyltransferase (puro^(r)), blasticidin S deaminase(bsr^(r)), xanthine/guanine phosphoribosyl transferase (gpt), or Herpessimplex virus thymidine kinase (HSV-tk).
 27. The mouse ES cell of claim21, wherein the one or more miRNA recognition sites are present in 1, 2,3, or 4 copies.
 28. The mouse ES cell of claim 21, wherein the one ormore miRNAs are selected from the group consisting of an miR 292-3p, anmiR 294, and a combination thereof.
 29. The mouse ES cell of claim 21,wherein the one or more miRNA recognition sites are present in 1, 2, 3,or 4 copies, and wherein the one or more miRNAs are selected from thegroup consisting of an miR 292-3p, an miR 294, and a combinationthereof.
 30. The mouse ES cell of claim 21, wherein the recombinase geneencodes a recombinase selected from the group consisting of Cre, Flp,and Dre.
 31. The mouse ES cell of claim 30, wherein the recombinase geneencodes a Cre recombinase, and the first and second recombinaserecognition sites are lox sites.
 32. The mouse ES cell of claim 21,further comprising a modified allele at a genomic locus of interest. 33.The mouse ES cell of claim 32, wherein the modified allele is notflanked by the first and second recombinase recognition sites.
 34. Themouse ES cell of claim 32, wherein the modified allele comprises aknockin, a knockout, a mutated nucleic acid sequence, or a combinationthereof.
 35. The mouse ES cell of claim 32, further comprising theselection cassette at the genomic locus of interest.
 36. A mouse embryocomprising the mouse embryonic stem (ES) cell of claim
 21. 37. The mouseembryo of claim 36, wherein the mouse ES cell further comprises areporter gene.
 38. The mouse ES cell of claim 37, wherein the reportergene is operably linked to the promoter of a targeted gene in the mouseES cell.
 39. The mouse embryo of claim 37, wherein the reporter gene isselected from the group consisting of a luciferase gene, a LacZ gene, aGFP gene, an eGFP gene, a CFP gene, a YFP gene, an eYFP gene, a BFPgene, an eBFP gene, a DsRed gene, and a MmGFP gene.
 40. The mouse embryoof claim 36, wherein the gene encoding the recombinase is flanked bythird and fourth recombinase recognition sites recognized by therecombinase.
 41. The mouse embryo of claim 36, wherein the selectioncassette comprises a gene that encodes neomycin phosphotransferase(neo^(r)), hygromycin B phosphotransferase (hyg^(r)),puromycin-N-acetyltransferase (puro^(r)), blasticidin S deaminase(bsr^(r)), xanthine/guanine phosphoribosyl transferase (gpt), or Herpessimplex virus thymidine kinase (HSV-tk).
 42. The mouse embryo of claim36, wherein the one or more miRNA recognition sites are present in 1, 2,3, or 4 copies.
 43. The mouse embryo of claim 36, wherein the one ormore miRNAs are selected from the group consisting of an miR 292-3p, anmiR 294, and a combination thereof.
 44. The mouse embryo of claim 36,wherein the one or more miRNA recognition sites are present in 1, 2, 3,or 4 copies, and wherein the one or more miRNAs are selected from thegroup consisting of an miR 292-3p, an miR 294, and a combinationthereof.
 45. The mouse embryo of claim 36, wherein the recombinase geneencodes a recombinase selected from the group consisting of Cre, Flp,and Dre.
 46. The mouse embryo of claim 45, wherein the recombinase geneencodes a Cre recombinase, and the first and second recombinaserecognition sites are lox sites.
 47. The mouse embryo of claim 36,wherein the mouse ES cell further comprises a modified allele at agenomic locus of interest.
 48. The mouse embryo of claim 47, wherein themodified allele is not flanked by the first and second recombinaserecognition sites.
 49. The mouse embryo of claim 47, wherein themodified allele comprises a knockin, a knockout, a mutated nucleic acidsequence, or a combination thereof.
 50. The mouse embryo of claim 47,wherein the mouse ES cell further comprises the selection cassette atthe genomic locus of interest.
 51. A method for making a geneticallymodified mouse, comprising: (a) introducing the mouse embryonic stem(ES) cell of claim 21 into a host mouse embryo, wherein the mouse EScell further comprises a modified allele at a genomic locus of interest,wherein the modified allele is not flanked by the first and secondrecombinase recognition sites; (b) implanting the host mouse embryocomprising the mouse ES cell of step (a) into a mouse capable ofgestating the host embryo; (c) maintaining the mouse of step (b) underconditions sufficient for gestation; and (d) obtaining from the mouse ofstep (c) a genetically modified mouse that lacks the selectable markerin its genome and comprises the modified allele.
 52. The method of claim51, wherein the mouse ES cell further comprises a reporter gene.
 53. Themethod of claim 52, wherein the reporter gene is operably linked to thepromoter of a targeted gene in the mouse ES cell.
 54. The method ofclaim 52, wherein the reporter gene is selected from the groupconsisting of a luciferase gene, a LacZ gene, a GFP gene, an eGFP gene,a CFP gene, a YFP gene, an eYFP gene, a BFP gene, an eBFP gene, a DsRedgene, and a MmGFP gene.
 55. The method of claim 51, wherein the geneencoding the recombinase is flanked by third and fourth recombinaserecognition sites recognized by the recombinase.
 56. The method of claim51, wherein the selection cassette comprises a gene that encodesneomycin phosphotransferase (neo^(r)), hygromycin B phosphotransferase(hyg^(r)), puromycin-N-acetyltransferase (puro^(r)), blasticidin Sdeaminase (bsr^(r)), xanthine/guanine phosphoribosyl transferase (gpt),or Herpes simplex virus thymidine kinase (HSV-tk).
 57. The method ofclaim 51, wherein the one or more miRNA recognition sites are present in1, 2, 3, or 4 copies.
 58. The method of claim 51, wherein the one ormore miRNAs are selected from the group consisting of an miR 292-3p, anmiR 294, and a combination thereof.
 59. The method of claim 51, whereinthe one or more miRNA recognition sites are present in 1, 2, 3, or 4copies, and wherein the one or more miRNAs are selected from the groupconsisting of an miR 292-3p, an miR 294, and a combination thereof. 60.The method of claim 51, wherein the recombinase gene encodes arecombinase selected from the group consisting of Cre, Flp, and Dre. 61.The method of claim 60, wherein the recombinase gene encodes a Crerecombinase, and the first and second recombinase recognition sites arelox sites.
 62. The method of claim 51, wherein the modified allelecomprises a knockin, a knockout, a mutated nucleic acid sequence, or acombination thereof.
 63. The method of claim 51, wherein the mouse EScell further comprises the selection cassette at the genomic locus ofinterest.
 64. A mouse made by the method of claim 51.